Loss of function of Vasoactive-intestinal peptide alters sex ratio and reduces male reproductive 1 fitness in zebrafish 2

Abstract


Introduction
Reproduction in vertebrates is regulated by the brain-pituitary-gonadal axis, in which internal and external cues are translated into endocrine signals that control various aspects of reproductive performance.The main axis is governed by the hypothalamic gonadotropin-releasing hormone (GnRH)     neurons that induce secretion of luteinizing-and follicle-stimulating hormone (LH and FSH, respectively) from the pituitary.LH and FSH, in turn, reach the gonads and regulate sex steroid synthesis and secretion 1 .Because GnRH was found to be dispensable for reproduction in zebrafish 2,3 , and based on transcriptomic analysis of WT and KO brains (unpublished), we examined Vasoactive intestinal peptide a (Vipa) as a potential factor regulating Lh in female zebrafish 4 .
Vip, a 28-amino acid neuropeptide, was initially isolated from the porcine gastrointestinal wall in 1972 5 .Vip is synthesized in various tissues, including the gut, pancreas, cortex, and suprachiasmatic nuclei (SCN) of the hypothalamus in the brain [6][7][8] .This pleiotropic neurohormone is known for its wideranging impacts such as stimulating contractility in the heart, inducing vasodilation, triggering glycogenolysis, reducing arterial blood pressure and relaxing the smooth muscle of the trachea, stomach and gallbladder 9 .
Vip plays a key role in male reproduction.Direct corpus cavernosum injection of Vip, alone or in combination with acetylcholine, has been shown to alleviate functional impotence through the stimulation of nitric oxide in mice 10,11 .In the testis, Vip is released near Leydig cell nests by specific Vip-ergic fibers that are abundant in the inferior spermatic nerve 12 .In fact, studies have reported the presence of Vip in axons penetrating the testis in various animals [12][13][14] .In vitro treatment of cultured Leydig cells with Vip resulted in dose-dependent increases in the production of testosterone, progesterone, and pregnenolone 15 , suggesting a direct stimulatory effect of Vip on Leydig cells.Vip-deficient males displayed lower FSH and testosterone levels in the serum, which is associated with early signs of testicular degeneration in mice 16 .
In mice, Vip neurons in the SCN innervate GnRH neurons in the median preoptic area (POA)     and convey circadian rhythm to the reproductive axis 17 .While the mechanism underlying Vip action on the testis in mammals is not fully clear, it was suggested that Vip originating from the SCN plays a role because circadian rhythm is perturbed in Vip null mice 18 .As a result, the rhythmic secretion of gonadotropins is disrupted in Vip KO mice and, in turn, so are testicular functions 18 .
Zebrafish possess two isoforms of Vip, Vipa and Vipb, in which Vipa peptide is most homologous to the mammalian Vip (e.g.mouse Vip shares 92% homology with zebrafishVipa).vipa gene was shown to be expressed in several brain regions of zebrafish, such as the anterior parvocellular preoptic nucleus (PPa), the postcommissural nucleus of the ventral telencephalic area (Vp), and the ventromedial thalamic nucleus (VM) of the hypothalamus 19 .Vipa neurons directly innervate Lh and Fsh gonadotropes in the pituitary of mature females and Vipa peptide intracerebroventricular (ICV) injection stimulated Lh secretion independent of Gnrh3 19 .Surprisingly however, this study also found that vipa -/-and vipa -/- /gnrh3 -/-mature ZF females displayed normal reproductive performance and were fertile.
In contrast, and for the first time in a teleost, we have found that reproductive fitness was profoundly and detrimentally affected by the lack of Vipa in vipa -/-males.In addition, sex ratio in vipa -/- offspring was biased towards females.Consequently, we set out to determine how the lack of Vipa Zebrafish male reproduction includes a typical mating behavior that precedes the sperm release.
During courtship, three initiatory mating activities (chase, tail-nose, and approach) are displayed by both males and females 20,21 .Both courtship and gamete quality are fundamental for successful spawning.
Spermatogenesis begins with differentiating germ cells that undergo transcriptional reprogramming and maturation through a diversity of cell types, with support of somatic cells 22,23 .Mammals and zebrafish share an overall testis architecture featuring several tightly coiled seminiferous tubules 24 .In the zebrafish testis, Sertoli cells surround individual undifferentiated spermatogonia, where germ cells first develop in a clonal syncytium of type A spermatogonia before undergoing mitotic divisions as type B spermatogonia and then subsequently entering meiosis 25 .This developmental sequence is followed by a spermiogenic phase, where spermatids develop into spermatozoa by nuclear condensation, organelle elimination, and formation of the flagellum.The cyst then opens to release mature spermatozoa in the lumen of seminiferous tubules 24 .Sertoli cells and Leydig cells produce testosterone and insulin-like growth factor 3 (Igf3), which promote spermatogenesis 26 27 .
In order to understand what causes sub-fertility in the absence of Vipa in male zebrafish, we examined local expression and presence of Vipa peptide in the testes of WT males and determined the effect of lack of Vipa on mating behavior, sperm potency, sperm quality and the androgen synthesis pathway.Our results suggest that endogenous testicular Vipa, which is expressed in developing spermatogonia and in Leydig cells, may also contribute to sperm quality, probably by regulating testosterone/11-KT producing enzymes.

Animal husbandry
All zebrafish (Tuebingen) were maintained in a 28°C recirculating aquaculture system with a 14hour light and 10-hour dark photoperiod.The Vipa -/-zebrafish line was previously established in our lab 19 .Before tissue sampling, fish were anesthetized to full sedation using tricaine methanesulfonate (MS-222; Sigma-Aldrich) and rapidly decapitated.The Institutional Animal Care and Use Committee at the University of Maryland School of Medicine approved all experimental protocols (institutional approval # 0519010 and #0522013).

Sex ratio
WT and vipa -/-zebrafish were in-crossed and offspring were raised to adulthood.After genotyping using PCR 19 , sex ratios from each spawn were determined for each genotype at 3 months of age based on external morphology.The sex of representative females and males was further verified through gonadal inspection and histology.

Masculinization of vipa -/-offspring
To test whether the female-biased sex ratio of the vipa -/-offspring can be rescued by testosterone, vipa -/-zebrafish larvae from four pairs were treated with 0.17 µM methyl-testosterone or ethanol vehicle in fish water for 15 days from 5 -20 dpf, following a previously published protocol 28 .The fish were reared in a spawning tank at 28 °C, with half of the incubation water volume replenished every other day.
At three months of age, the sex of each individual was determined based on external morphologies and

In vitro fertilization
WT female and WT or vipa -/-males were paired for spawning the night before, following routine procedure.Sperm (1-2 µl) was collected in the morning by gently stroking the sides of the fish around the urogenital region between the thumb and forefinger, followed by gentle suction with a 2 µl pipette at the urogenital opening, and stored in 50 µl Hank's Balanced Salt Solution on ice.Subsequently, eggs were collected by gentle stripping from WT females.The sperm from each vipa -/-and WT males were added to the same number of oocytes from eight different WT females in separate 60 mm culture plates.Five milliliters of fish water were added, and the fertilization process was initiated at 28°C with gentle agitation at 30 rpm.Fertilization rates were quantified after 24 hours by counting fertilized and unfertilized eggs.

Fertilization potency
5-month-old vipa -/-and WT siblings were mated with the following combinations: 14 vipa -/-males and 10 vipa -/-females with WT counterpart and WT males with WT females.In each of the mating combinations, one female and one male were set for spawning in standard zebrafish spawning tanks with the sexes separated by a divider, which was removed immediately after lights-on at 09:00 h for a 2-hour period to incite mating.Eggs were collected from each breeding chamber and incubated in fish water at 28 o C. Fecundity (spawned eggs/body weight) and fertilization rate (number of fertilized eggs/total eggs oviposited) were quantified by counting fertilized and unfertilized eggs at 24 hpf, survival rate was

Male to female attraction
Male vipa -/-and WT approaches toward a female were tracked.Six males of each genotype group were used for the assay.To minimize variations, all animals are age-and size-matched at 4 months.A male from either genotype, along with a WT female, were placed in the same chamber, separated by a plastic center divider 20 hours before the actual assay.The next morning, the pairs were transferred to a modified container with a narrow compartment that limited fish movement (3 cm) holding a WT female or male (as a control for sexual attraction).The tested male was placed in the larger 20 cm compartment, which allows free swimming.Immediately after transferring the animals to the assay chamber, the tested male behavior was recorded for 10 min by a GoPro Hero 8 camera.The number of male approaches towards the divider was counted, and all movements were tracked using idTracker software 29 .

Sperm quality
WT or vipa -/-males were anaesthetized with MS-222.The genital area of the male was gently pat-dried before 1-2 µl sperm was collected directly from the genital opening using a 2 µl pipette tip and transferred into 50 μl Hank's solution and stored on ice.All sperm assays were performed within 15 min after the collection.The diluted sperm was activated by adding 50 μl fish water at room temperature.Then 5 μl activated sperm solution was then loaded on a semen analysis slide with a sperm counting grid (10 µl w/ Grid -Disposable Makler, CellVision) and immediately filmed for 10 minutes using the Olympus BX60 Fluorescence Microscope and Volocity 6.2.1 system, followed by analysis with Volocity 6.2.1 software (https://www.volocity4d.com)and CASA software (https://github.com/calquezar/OpenCASA) 30.

Gonadal histology via H&E staining
Testes were sampled by dissection from sexually mature WT and vipa -/-males at 5 months of age and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C overnight.The testes were transferred into glass vials containing 30% sucrose in 0.1 M phosphate buffer at 4°C and allowed to completely submerge.After mounting with optimum cutting temperature (OCT) resin (Sakura Fineteck, Inc.), the testes were sectioned sagittal at 5 µm thickness using a cryostat (Sakura Fineteck, Inc.), and sections were transferred to positively charged slides (Denville UltraClear; Denville Scientific).
The sections were post-fixed with 4% PFA and stained with hematoxylin and eosin (H&E) following a standard protocol, dehydrated, mounted and viewed under Zeiss Axioplan microscope (Carl Zeiss Microscopy).

In situ hybridization
Slides for ISH were prepared similar to H&E staining except for preparing 10 µm thick sections.The slides were post-fixed with 4% PFA, incubated with 1 µg/mL proteinase K (New England Biolabs) in PBS at 37 °C for 10 minutes and post-fixed with 4% PFA in PBS at room temperature for 20 minutes.
Vipa DIG labeled anti-sense and sense riboprobes were prepared using vipa cDNA as a template (GenBank accession # NM_001114553.3)and T7 or Sp6 RNA polymerase.The sections were prehybridized at 62 °C for 2 hours followed by a hybridization with 1000 ng/mL of a generated vipa antisense or sense DIG-labeled riboprobe overnight at 62°C.After washing, sections were blocked with TNB blocking buffer (100 mM Tris-HCl, pH 7.5; 0.15 M NaCl; 0.5% NBT blocking reagent, from Tyramide Signal Amplification kit, TSA; Perkin Elmer).Slides were incubated with anti-DIG Fab Roche).After mounting and counterstaining with nuclear red solution (0.1% nuclear fast red, 5% aluminum sulfate), the slides were imaged using a Zeiss Axioplan microscope (Carl Zeiss Microscopy).

Immunohistochemistry
IHC was performed on sagittal testes sections (5 micron paraffin sections) from 5 -month-old sexually mature WT and vipa -/-males.After de-paraffinization and rehydration, slides were immersed 15 minutes in 0.1 M citrate buffer, pH 6.0, in a 95 o C water bath for antigen retrieval.Endogenous peroxidases were inactivated with 0.5% H 2 O 2 in 1x PBS for 30 min at RT, and nonspecific background was reduced by incubation in normal goat serum with BSA for 1 hour at room temperature.Sections were then incubated overnight at 4°C with anti-zfVIPa primary antibodies (raised against the recombinant Vipa precursor, Zohar Yonathan -UMBC Cat# Zohar Lab, RRID:AB_3101965) diluted 1:1000 in PBST containing 2% normal goat serum as previously described 4 , washed and incubated with goat anti-rabbit antibody conjugated to peroxidase (Perkin Elmer).The signal was revealed using 3,3 -diaminobenzidine as the chromogen.Sections were counterstained with hematoxylin solution (Sigma Aldrich).Negative controls were carried out using the pre-immune serum from the same immunized rabbits.

Real-time qRT-PCR and 11 -ketotestosterone ELISA on testes
qRT-PCR-Total RNA was extracted from testes of 5-month-old vipa -/-and vipa +/-male siblings using RNAzol reagent, according to the manufacturer's protocol, and total RNA was quantified with a Nanodrop unit (Thermo Scientific).One μg total RNA was treated with RQ DNAse 1 at 37°C and 11β-hydroxylase (cyp11b1) in the testis were determined by amplification using primer sets described elsewhere 31 32 with eef1α as an internal control (Table 1).mRNA levels were normalized using a datadriven normalization algorithm (NORMA-Gene) method 33 .The normalization was performed using seven genes from the same tissue.The algorithm estimates a normalization factor by calculating mean expression values for each replicate for all genes.For each sample, duplicate data is obtained and averaged prior to normalization, then the fold-change in normalized expression relative to the control was calculated.Biological replicates were averaged to obtain mean fold-change in gene expression ± standard error of mean (SEM) as previously described 34 .
11-KT ELISA-Seven testes were dissected from 3-month-old vipa -/-and WT males, respectively and gonad weight was recorded.Steroid extraction followed the protocol described elsewhere 35 .Briefly, the testes were incubated separately in 5 ml 80% methanol at 4℃ for at least 48 hours in low-protein binding tubes (Eppendorf).Testes were then sonicated using a 'Branson Sonifier 250' sonicator equipped with wide probe for 30 sec at output level 5, centrifuged at 12,000 rpm for 15 min at 4℃ and the supernatant was transferred to a new low protein-binding tube.One ml aliquot of the resulting supernatant was dried in a Speedvac for two hours, and then reconstituted with1 ml of ELISA steroid assay buffer.

Vipa knockout males display reduced fecundity and fertilization capacity
To determine the reproductive fitness of vipa -/-zebrafish male, vipa -/-males or female zebrafish were crossed with WT counterparts followed by fertility and fecundity tests.The total eggs obtained from 11 pairs of vipa -/-females crossed with WT males was 5925, and 15 pairs of vipa -/-males crossed with WT females was 5159.The mean fertilization rate for each group: vipa -/-female with WT male was 84.18 ± 3.286% and vipa -/-male with WT female was 27.73 ± 3.811% (P <0.0001), fecundities of 538 ± 57 and 343 ± 37 on average per female, respectively (p= 0.0069) (Figure 1 A-C).
To determine whether the low fecundity and fertilization rate were caused by compromised mating behavior, we also performed in vitro fertilization trials, where sperm of vipa -/-and WT males was used to fertilize eggs obtained from the same WT female and divided equally.Significantly lower fertilization rates (28.83 ± 10.41%) were recorded for vipa -/-sperm compared to WT sperm (90.43 ± 1.28%) (p = 0.0001), or compared to vipa -/-eggs fertilized with WT sperm (94.74 ± 2.09%) (p= 0.001383) (Figure 1 D).

Sex ratio is female-biased in vipa -/-offspring and is reversed with methyl-testosterone
The sex ratio of female to male of vipa -/-was consistently biased towards females with an average 81.5 ± 1.06 % to 18.5 ± 1.09 % female-to-male ratio, compared to a 57 ± 3.39 % and 43 ± 3.3 % ratio in WT.All females with female external morphologies had ovaries.To understand whether female -biased sex ratio in vipa -/-could be reversed by exposure to testosterone, the offspring were subjected to a masculinization procedure using methyl-testosterone.The treatment increased the male prevalence to 74.16 ± 3.27 % and 90.16±1.61%and reduced that of females to 25.84 ± 3.27 % and 9.84±3.27%for vipa -/-and WT, respectively (Table 2).

Vipa -/-males exhibit reduced attraction to females
To investigate the effect of Vipa on male zebrafish sexual and motivation, we tracked the swimming pattern of WT and vipa -/-male zebrafish with similarly sized WT female zebrafish or WT male.In general, vipa -/-males exhibited less approaches towards the confined fish (described in Methods), regardless of the sex, than WT males.Vipa -/-males spent an average of 1.26 ± 0.35 mins, while WT males spent an average of 4.03 ± 1.02 mins of the recorded 10 mins within 3 cm distance of the WT female chamber (Figure 2A) (p<0.0001).On average, the mean distance from the confined female exhibited by WT and vipa -/-was 53 and 94 mm, respectively.This distance grew to 85 and 103 mm, respectively, when a male was placed in the confinement chamber.During the 10 min recording, vipa -/- males wandered away from the divider, while WT males often swam into a 30 mm range of the divider (Figure 2 A,B).

Vipa -/-males exhibit smaller and disorganized testis and reduced sperm quality
To determine the effect of lack of Vipa on the testis, we examined testis morphology and sperm quality.
Gross morphology (Fig. 3 A,B) and GSI (Fig. 3 F) analysis of testes from WT and vipa -/-males revealed significantly (2 times) smaller testes in vipa -/-compared to WT. Histological inspection of testicular sections with H&E staining revealed a striking difference between the two genotypes: while WT testis contained a large number of mature spermatozoa (Fig. 3E), that of vipa -/-contained significantly less spermatids with empty spaces throughout the tissue (Fig. 3C and D).Sperm quality of WT and vipa -/-males was determined by both sperm count and motility using the CASA algorithm 30 .Sperm concentration of WT averaged ~1000/µl while that of vipa -/-males was significantly (50%) lower, averaging ~500/µL (Fig. 4A).
Progressive motility (rapid straight or large circles) (Fig. 4B) and non -progressive motility (small tight circles) (Fig. 4C) of vipa -/-males lasted about half and one-third the time of WT males, respectively.

Vipa mRNA and peptide are detected in testis
In light of the profound effect on the testis, we sought to test whether vipa is endogenously expressed in the zebrafish testis.Therefore, we examined the presence of vipa mRNA using ISH and Vipa peptide via IHC in the testes of WT and vipa -/-zebrafish.vipa mRNA was detected in Leydig cells, spermatocytes A and B, spermatogonia and spermatids (Fig. 5B).Vipa peptide was detected in pre -spermatogonia, primary spermatocytes, second spermatocytes, Leydig cells, and Sertoli cells in the WT zebrafish testis (Fig. 5D, E).Immuno-staining of vipa -/-testis with the same antibodies did not result in any signal (Fig. 5F).Immuno-staining using the pre-immune serum did not result in signal on consecutive sections (Fig. 5G).
To determine the effect of lack of Vipa on 11-Ketotestosterone levels, we measured the 11-KT in the testes of WT and vipa -/-.Levels of 11-KT in WT testes varied from ~250 to 600 pg/mg testis tissue between the 7 individuals averaging ~400 pg/mg.11-KT levels in vipa -/-testes were undetectable in 4 out of the 7 testes and close to 0 in the other three (Fig. 6B)

Discussion
In the framework of this study, we demonstrated that Vipa-deficient mature zebrafish males are subfertile, indicating that Vipa has an important functional role in the testis.Through a series of tests, we show that sperm quality, potency and attraction to females are severely compromised in the vipa -/-males.We first noticed the sub-fertility condition during efforts to propagate the vipa -/-line.Our attempts to incross homozygous mutant fish were largely unsuccessful.Further examinations, which include different combinations of crosses of heterozygous and homozygous vipa -/-with WT males and females substantiated our initial observations.In addition, we have encountered great difficulty obtaining a sufficient number of males from the few small spawns obtained from vipa -/-parents.While Dmrt1 (double-sex-and mab-3-related transcription factor 1) and anti-müllerian hormone (AMH) that promotes male sexual differentiation 39,40 are established factors, information about the role of Vip in sexual differentiation is scarce.However, one study implicated Vip in sexual differentiation of male mice neonates through the mediation of prolactin response to androgens 41 .In contrast, to date, there is no strong evidence for the involvement of androgens in sex differentiation in fish 42 , including in zebrafish 43 .In fact, blocking of endogenous estrogen synthesis in the ovaries induces complete sex reversal to fertile males [43][44][45] .In that regard, it has been shown that administration of methyl testosterone suppresses the expression of all steroidogenic enzyme genes in vitro, including cyp19a1a aromatase, which converts testosterone to estradiol 46 .Hence, it is possible that the downregulation of cyp19a1a is the reason for the masculinization of both WT and vipa -/-fingerling observed in our study.Nevertheless, despite our finding that the expression of critical androgenic enzymes and 11-KT levels are reduced in mature vipa -/-males, the mechanism underlying Vipa involvement in sex differentiation has yet to be determined.
Vip mRNA levels and distribution in the brain and pituitary display sexual dimorphism.In mammals, including in humans, Vip levels in certain brain regions are higher in males 47,48 , and in the pituitary this dimorphism is associated with the prolactin secretory effect of Vip 49 .Interestingly, in the medaka, Vip expression in neurons populating a defined nucleus in the POA also innervate the pituitary in females, while pituitary adenylate cyclase-activating polypeptide (Pacap) observed in the same nucleus is found in males 50 .Because fshβ and lhβ double mutant zebrafish are all males, with delayed testicular development 51 , the possibility that the lack of Vipa in the vipa -/-male hinders testicular development through its effect on gonadotropins cannot be ruled out.Because our study determined that Vipa is expressed in the testis, Vipa may endogenously regulate testicular function, suggesting a dual regulatory pathway by VIPa.The possibility of a dual brain-pituitary and gonadal regulation by Vip is supported by the fact that VIP is found and exerts its control both at the hypothalamic-hypophyseal level, as well as in the genital organs in mammals 52 .Nevertheless, the observation that female zebrafish reproduction is not affected by the lack of Vipa is surprising because the importance of VIP to the function of the ovary is widely documented 53 .This may infer that brain-pituitary Vip plays a minor role in the regulation of the gonads in zebrafish.
SCN Vip is one of the two major neuropeptides mediating circadian rhythms in mammalian brain.SCN Vip neurons directly innervate GnRH neurons and mediate their activity 54 .Unlike vertebrates, teleost brain lacks SCN and circadian rhythm is controlled in a decentralized fashion, with all tissues and the majority of cells possessing a direct-light entrainable circadian pacemaker 55 56,57 .Hence, Vipa may function as a circadian clock regulator in the many tissues that express it.Further studies are required to test this possibility.
Vipa mRNA and protein are found in the testicular tissue of mature zebrafish males, including in Leydig cells, where steroidogenesis takes place, but also in developing and mature spermatids.Examination of the published transcriptome data from bulk WT zebrafish testis detected transcripts of vipa and genes encoding its cognate receptors, i.e. adcyap1b, vipr1b, vipr2, vpac2r and adcyap1r1a (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM5820552) 58 .In mammalian vertebrates, Vip is found in nerves surrounding and penetrating the testis of mice 12,15,59 , where it induces steroidogenesis.Despite efforts, we could not co-localize Vipa peptide in nerve structures in the testis, as described in mammalian species.Congruently, the presence of Vip was previously shown in mitotic and differentiating germ cells as well as in Leydig, Sertoli cells, prespermatogonia and spermatogonia of two non-mammalian vertebrates, the cartilaginous fish Torpedo marmorata and the wall lizard Podarcis sicula, 60,61 .These differences in the testicular expression pattern of vipa between mammalian and non-mammalian vertebrates may represent evolutionary projection of this gene, i.e. from broad expression in the ray (Elasmobranchii), to a limited expression in zebrafish (Cypriniformes) and no endogenous expression in mammalian species.
We have established that vipa -/-male subfertility is caused by poor sperm quality characterized by lower sperm count, reduced motility range and span, as well as lack of attraction to females.vipa -/-males displayed a pronounced reduction in their attraction to females compared to WT males, but surprisingly also slightly lower attraction when compared to vipa -/-male to WT female or WT male.This may suggest that the absence of Vipa affects social behavior to a lesser degree.
Both sexual behavior and sperm quality are highly dependent on testosterone and 11-ketotestosterone (11-KT) levels 62 .Although the expression levels of the steroid synthesis pathway-related genes tested in the heterozygous vipa -/+ males were highly variable between individuals, these variations were significantly reduced in the vipa -/-cohort.This pattern of high diversity of gene expression in the testis has been described in other studies 63,64 , and is probably a common phenomenon for some genes.Indeed, we have found that gene expressions of 3β-hsd, 17β-hsd1 and 11β hydroxylase are downregulated in vipa -/-testis, which implies that levels of testosterone, dihydroxytestosterone and 11-KT may, in turn, be depleted 65 .Congruently, and unlike in WT testes, 11-KT levels in the vipa -/-testes were significantly reduced and were often undetectable, thus supporting the idea that androgen synthesis is compromised in vipa -/-testis.Of the three enzymes, 17β-hsd1, which converts androstenedione to T, is required for male mouse fertility 66 , whereas the essentiality of 3β-hsd, which catalyzes the biosynthesis of progesterone from pregnenolone, 17 α-hydroxyprogesterone from 17α-hydroxypregnenolone, and androstenedione from dehydroepiandrosterone (DHEA), in male reproduction is controversial 67 .Moreover, 11 β-hydroxylase and ferredoxin 1β male mutant zebrafish, the latter of which is required for androgen synthesis, exhibited secondary female sex characteristics associated with lower androgen levels 32,64 .Unlike vipa -/-, these males had viable sperm when tested in vitro.However, in contrast to the findings in (30, 52), we have found that all vipa -/-females had only ovaries, implicating Vipa in the development of male secondary sex characteristics.The differences between lower androgen levels due to 11 β-hydroxylase and ferredoxin 1b deficiency, which directly impact androgen levels, and the Vipa mutant, which also features Vipa deficiency, suggest that Vipa has an additional non-steroidogenic effect in the testis.
As added support, incubation of human sperm with the VIP peptide increased its motility and the concentration of motile sperm beyond the steroid-dependent phase 68 .The likelihood that Vipa plays several roles in the testis is supported by the presence of Vipa not only in the steroidogenic Leydig cells, but also developing and mature spermatids (at various stages of development).Interestingly, androgen receptor (ar) deficient zebrafish males are strikingly similar phenotypically to the vipa -/-male (e.g.smaller testis size, infertility when tested by natural mating, and only a small amount of mature spermatozoa with a lower fertilization capacity by in vitro fertilization) 69 .This suggests that loss of function of a single androgen synthesis gene results in a moderate response compared to the more dramatic disruption caused by the inactivation of the androgen receptor.When taken collectively, the information above signifies that Vipa has a comparable importance to that of the androgen receptor.
This study demonstrates the importance of Vipa to male reproduction and male sex differentiation in zebrafish.We showed that lack of Vipa results in downregulation of three major enzymes in the synthesis chain of androgens in the testis, which may in turn decrease testosterone production.The putative lower testosterone and 11-ketotestosterone levels may explain the biased sex ratio, as well as the underdeveloped testes and lack of sexual motivation in vipa -/-males.However, because lower androgen levels do not hamper sperm quality, Vipa may contribute to testicular function via other pathways.Further studies are required to determine whether the source of Vipa is solely the testis or a combination of sources that includes brain/circulation sources and testicular production.Table 2: Sex is biased towards females in vipa -/-offspring and is reversed with androgen treatment.S ex ratio of homozygous vipa -/-and WT offspring treated with methyl-testosterone or only vehicle.Sex wa s determine in 3 month-old adults.Statistical analysis performed using One Way ANOVA, followed by T ukey's Multiple Comparisons Test. Figure 2: vipa -/-males display reduced attraction to female.vipa -/-and WT males' distance from WT female and male zebrafish and the time spent near confined females or males.A) Time spent within 30 mm of female/male chambers by vipa -/-and WT males (n=5 males of each type resepectively).B) Tracking distribution map of a representative male of each type (WT and vipa -/-) using idTracker software to assess images taken at 30 frames/sec intervals.C) 30 sec representative tracking videos of a vipa -/-(left) and WT male (right) with the same confined WT female (circled).Statistical analysis for (A) was performed on mean values using One Way ANOVA, followed by Tukey's Multiple Comparisons Test, ****= p < 0.0001.Figure 3. Gonadal morphology and gametogenesis of vipa -/-and WT testes.Gross testes morphology (in situ) of two representative vipa -/-and WT adult males (A; top row of images), and after excision (B; bottom row of images).(C) Testis histology with hematoxylin and eosin staining of vehicle treated vipa -/- male, (C) Testis histology with hematoxylin and eosin staining of methyl-testosterone treated vipa -/-male, (E) Testis histology with hematoxylin and eosin staining of WT male.(F) Average GSI of WT and vipa -/- males (n=6).Statistical analysis performed using the Welch Two Sample T-test.P (GSI)= 0.0092 and 0.0002, **=p < 0.01, ***=p < 0.001.
Funding: NSF-BSF #1947541: Studying the compensatory mechanisms underlying gene loss-of-function in the nervous system.

Figure 5 :
Figure 5: Vipa mRNA and peptide are detected in zebrafish testis.In situ hybridization using vipa

Figure 6 :
Figure 6: The effect of loss of vipa on androgen synthesis and sex steroid hormone levels.(A) Downloaded from https://academic.